Analysis of Drugs of Abuse by GC/MS Using Ultra Inert Universal ...

Application Note Forensics

Analysis of Drugs of Abuse by GC/MS Using Ultra Inert Universal Sintered Frit Liners

Author

Angela Smith Henry, Ph.D. Agilent Technologies, Inc.

Abstract

Gas chromatography/mass spectrometry (GC/MS) is a common screening technique for controlled substance analyses. Maintaining an inert flowpath is important in these analyses to prevent loss of peak shape and signal of the more sensitive or active compounds, such as amphetamine or oxycodone. A sintered frit liner offers the same protection from complex nonvolatile matrices as glass wool liners, while avoiding wool breakage that may cause loss of peak response.

Introduction

Maintaining a clean and inert GC/MS system starts at the inlet with an inert flowpath, specifically with the inlet liner. Using deactivated liners provides a good start for preventing peak degradation in the inlet. Inlet liners with glass wool are used because the wool provides a large surface area to aid in sample vaporization. Wool also provides a barrier to trap nonvolatile residue from sample matrices.1,2 However, glass wool in liners can re-introduce active sites over the lifetime of the liner that manifest as a decrease in peak response or degradation of peak shape. A sintered frit liner provides the surface area for vaporization, and can reduce the sample loss by preventing sample droplets from reaching the bottom of the inlet before vaporization. Fritted liners also provide the ability to trap nonvolatile residue, while removing the possibility of new active sites from broken glass wool, or the intrusion of glass wool into the head of the column.

GC/MS screening methods are important for laboratories running controlled substance analyses, as new illicit substances continue to enter the marketplace, and a target compound list can stretch into the hundreds. For compounds compatible with GC, GC/MS can be used in full scan mode with electron ionization (EI) mode to complete the controlled substance screening. Some compounds, such as amphetamines, can be very sensitive to inlet parameters, inlet liners, and the solvent for dilution. These amphetamines may exhibit bad peak shape if the inlet liner and parameters are not optimized. Improving amphetamine peak shape and good peak response remain important factors in liner technologies. Powder samples are generally dissolved in a

solvent such as methanol, hexane, or toluene, while liquid samples are extracted into GC-amenable solvents, and possibly diluted to avoid overloading the GC column or MS detector. Liners were tested with the Agilent forensic toxicology checkout mixture, which includes compounds from different classes such as amphetamines, opiates, and benzodiazepines, along with other high-concentration mixtures of amphetamines, opiates, and cannabinoids.

Experimental

Chemicals and reagents

The Agilent GC/MS forensic toxicology checkout mixture standard (p/n 51900471, 5 g/mL) was used to test the liners. Table 1 lists the compounds found in the mixture with the retention times. HPLC/GC grade toluene and methanol were purchased from MilliporeSigma (Burlington, MA, USA). An internal standard (ISTD) mixture of six deuterated polyaromatic hydrocarbons (PAHs) was purchased, containing 2 mg/mL of 1,4-dichlorobenzene-d4, acenaphthene-d10, naphthalene-d8, phenanthrene-d10, chrysense-d12, and perylene-d12 in acetone. The 5 g/mL checkout mixtures had 1 ?L of ISTDs added per 1 mL of the checkout mixture for a final concentration of 2 g/mL for the ISTDs.

Multiple mixtures were purchased from Cerilliant Corporation (Round Rock, TX, USA) and from Cayman Chemical (Ann Arbor, MI, USA) to test peak shape integrity with the sintered frit liner. The multicomponent opiate mixture contained methadone, codeine, hydrocodone, meperidine, and oxycodone each at 250 g/mL. The amine mixture contained amphetamine, methylenedioxyethylamphetamine

(MDEA), methamphetamine, methylenedioxymethamphetamine (MDMA), Methylenedioxyamphetamine (MDA), and phentermine, each at 250 ?g/mL. The cannabinoid mixture contained cannabidiol, cannabinol, and tetrahydrocannabinol (9-THC) at concentrations of 1.0 mg/mL. GC/MS drug mixture 1 contained varied concentrations of caffeine (40 g/mL), methadone (30 g/mL), cocaine (30 g/mL), codeine (50 g/mL), 6-monoacetylmorphine (75 g/mL), and heroin (75 g/mL). GC/MS drug mixture 3 contained methamphetamine, cocaine, heroin, fentanyl, and alprazolam at concentrations of 1.0 mg/mL.

An acetaminophen caplet was used to simulate a real-world matrix. The acetaminophen caplet was crushed and dissolved in a 25% methanol/75% water mixture. The methanol/water/ acetaminophen mixture was then extracted into toluene for GC injection.

Instrument conditions

An Agilent 7890 GC was connected to an Agilent 5977B Inert Plus GC/MSD with a 9 mm extraction lens. The GC was also equipped with an Agilent 7650A automatic liquid sampler, which has the larger turret to hold up to 50 samples. A 20 m ? 0.18 mm ? 0.18 ?m column was used to increase the system efficiency of the analysis with a split 20:1 injection, as controlled substance samples tend to be at higher concentrations. This method has been used for other forensic drug screening applications.3 The Agilent universal single taper frit liner (p/n 5190-5105) was chosen as the primary test case. The Inert Plus MSD was run in scan mode with extraction tune (etune.u). Table 2 lists the GC and MSD instrumentation and consumables, and Table 3 lists the method parameters.

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Table 1. Agilent GC/MS forensic toxicology checkout mixture compounds and retention times in elution order.

No.

Compound

1

Amphetamine

2

Phentermine

3

Methamphetamine

4

Nicotine

5

MDA

6

MDMA

7

MDEA

8

Meperidine

9

Phencyclidine

10 Methadone

11 Cocaine

12 Proadifen

13 Oxazepam

14 Codeine

Retention Time (min) 1.535 1.684 1.758 2.536 3.272 3.563 3.818 4.803 5.602 6.762 7.09 7.556 7.678 7.948

No.

Compound

15 Lorazepam

16 Diazepam

17 Hydrocodone

18 THC

19 Oxycodone

20 Temazepam

21 Flunitrazepam

22 Heroin

23 Nitrazepam

24 Clonazepam

25 Alprazolam

26 Verapamil

27 Strychnine

28 Trazadone

Retention Time (min) 8.017 8.144 8.213 8.276 8.531 8.758 8.832 8.896 9.441 9.748 10.177 11.231 11.358 12.666

Table 2. GC and MSD instrumentation and consumables.

Parameter GC MS Drawout Plate Column Liner Inlet Septum Autosampler Vials Vial Inserts Vial Screw Caps Test Mixture

Value Agilent 7890 GC Agilent 5977B Inert Plus GC/MSD with inert EI source 9 mm (p/n G3870-20449) Agilent DB-5ms Ultra Inert, 20 m ? 0.18 mm ? 0.18 ?m (p/n 121-5522) Agilent universal single taper with sintered frit (p/n 5190-5105) Agilent Advanced Green, nonstick 11 mm septum (p/n 5183-4759 for 50 pack) Agilent 7650A automatic liquid sampler Agilent A-Line certified amber (screw top) vials; 100/pk (p/n 5190-9590) Agilent deactivated vial inserts; 100/pk (p/n 5181-8872) Agilent screw caps, PTFE/silicone/PTFE septa, cap size: 12 mm; 500/pk (p/n 5185-5862) Agilent GC/MS forensic toxicology checkout mixture standard, 5 g/mL (p/n 5190-0471)

Table 3. GC and MSD instrument conditions.

Parameter Injection Volume

Inlet

Column Temperature Program Carrier Gas and Flow Rate Transfer Line Temperature Mode Ion Source Temperature Quadrupole Temperature Mass Range A/D Samples Tune

Value 1 ?L Split/splitless inlet 250 ?C; Split 20:1; Standard septum purge (3 mL/min) 110 ?C, 20 ?C/min to 300 ?C (hold 4.5 minutes) Helium at 1.5 mL/min, constant flow 280 ?C Scan 250 ?C 150 ?C m/z 40 to 500 4 Etune.u

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Results and discussion

This testing evaluated the Ultra Inert sintered frit liners for screening of controlled substances by GC/MS. The ability of the liners was determined by chromatographic evaluation, linertoliner reproducibility, and response repeatability across multiple injections on the same liner.

Chromatographic performance and amphetamine peak shape

The adsorption or decomposition of basic drug compounds may cause a variety of chromatographic issues such as distorted peak shapes, broad or tailing peaks, or loss in response and sensitivity. Mixtures of individual compound classes, specifically amines, opiates, and cannabinoids were tested for these chromatographic issues before moving onto samples with mixed compound classes. Peak shape issues or loss of sensitivity commonly occur for the early-eluting amine compounds such as amphetamine, methamphetamine, phentermine, and MDA. This method was designed for a high-efficiency column for better results, where the method was optimized for peak shape of the amines.3 Figure 1 illustrates the optimization of the method for the early-eluting amine peaks. The amphetamine mixture was injected neat, at 250 g/mL, with the 20:1 split lowering the concentration to 12.5 g/mL on-column. This highconcentration sample was used, since controlled substance testing tends

to have higher concentration samples. The amine compounds are well resolved with well shaped peaks in both the total ion chromatogram (TIC) and extracted ion chromatograms (EICs) (Figure 1A and 1B). The amines mixture was also

diluted with toluene to a final analyte concentration of 10 g/mL. An injected sample of 10 g/mL results in 0.5 g/mL on-column, and is used to verify peak shape integrity at lower concentrations and system inertness.

A 23

1

1. Amphetamine 2. Phentermine 3. Methamphetamine 4. MDA 5. MDMA 6. MDEA

6 5 4

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min)

B

3

2

1

1. Amphetamine 2. Phentermine 3. Methamphetamine 4. MDA 5. MDMA 6. MDEA

5 44 m/z 58 m/z 91 m/z 136 m/z

4

6

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 Time (min)

Figure 1. A) TIC of amphetamine mixture at 250 g/mL (split 20:1 for oncolumn concentration of 12.5 g/mL); B) extracted ion chromatograms (EICs) of amphetamine mixture at 250 g/mL.

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As shown in Figure 2, the EICs for the amine compounds at 10 ppm retain similar peak shape and responses as the higher concentration results, indicating that the method, liner, and flowpath are optimized and inert.

Peak shapes and resolution of other compound classes were also evaluated with additional standard mixtures, including opiates and cannabinoids. The opiates mixture was tested at neat concentration (250 g/mL injected), and with a 10 g/mL sample, diluted in toluene. Hydrocodone and oxycodone can be sensitive indicators for activity in the flowpath. If the column is degrading or the inlet and liner have active sites or are becoming dirty, the peak heights will diminish, and for oxycodone, a significantly broad, tailing peak will develop. Viewing Figure 3A, the high-concentration standard (250 g/mL) shows excellent separation and response for these opiates. There is minor tailing across all compounds. However, injection of the 10 g/mL sample (Figure 3B) illustrates less tailing, indicating that the tails are related to the concentration on-column rather than flowpath activity. The cannabinoids standard was diluted to 10 g/mL in toluene. Similar to the other compound class mixtures, the cannabinoids are well resolved from each other and have excellent peak shape, as observed in Figure 4.

23 1

1. Amphetamine 2. Phentermine 3. Methamphetamine 4. MDA 5. MDMA 6. MDEA

5

44 m/z

58 m/z

91 m/z

136 m/z

4 6

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min)

Figure 2. EICs of amphetamine mixture at 10 g/mL (split 20:1 for on-column concentration of 0.5 g/mL).

A

Meperidine

Methadone

Hydrocodone Codeine

Oxycodone

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5 min

B

Meperidine

Methadone

Hydrocodone

Codeine Oxycodone

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

min

Figure 3. TICs of opiates mixture at A) 250 g/mL, which is split 20:1 for oncolumn concentration of 12.5 g/mL and at B) 10 g/mL, split for on-column concentration of 0.5 g/mL.

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